During fiscal year 2010 we accomplished the following: 1) Completed and published studies of Fanconia anemia complex association with mononucleosomes reconstituted in vitro. 2) Utilized a novel biochemical method to prepare and purify histone H3 that is trimethylated at position 4 (H3K4me3). The method consists of producing a mutated H3 that contains a cysteine residue instead of lysine at position 4. Purified H3C4 is alkylated with (2-bromoethyl)trimethyl ammonium bromide to produce a H3 derivative that is closely related to H3K4me3. This mutant histone was reconstituted into octamers assembled with H2a, 2b and H4 expressed and purified from bacteria. Since bacterially expressed histones lack any other post-translational modifications, the resulting octamers are exclusively modified at position 4 of histone H3. 3) We used assembled octamers that contain modified or unmodified H3 to assemble mononucleosomes with a DNA sequence that contains a RAG1/2 recognizing recombination signal sequence. These substrates were treated with RAG1/2 complexes that contain full-length RAG2 (that recognized H3K4me3) or core RAG2 (that lacks the PHD domain that binds H3K4me3). Preliminary results show that the DNA associated with H3K4me3-containing mononucleosomes is cleaved better with full length RAG2. These studies are being done in collaboration with Dr. Michael Lieber (University of Southern California). 4) We assembled plasmid substrates that contain 12- and 23- RSSs into chromatin using unmodified nucleosomes or those that contain the specific modification described above. We purified full-length RAG2 protein and core RAG1 protein for further studies of coupled cleavage of 12- and 23-RSS- containing plasmids.